Initiation of DNA replication is the most important step in the regulation of this process since it ensures that it occurs during the S phase of the cell cycle and that all DNA regions replicate precisely only once. Initiation depends on the activation of DNA regions known as origins. Defining the DNA elements required for origin activity is central to our understanding of control of cell division in normal and disease states.This information is lacking in metazoan organisms. Thus, the questions as to which DNA elements play a role in the initiation step of DNA replication in mammalian cells, and whether unique regions are utilized, remain to be answered. Our previous work has led to the isolation and cloning of human DNA fragments that remain associated to the nuclear matrix after restriction enzyme digestion of HeLa nucleoids. We showed that this DNA population was enriched for forked DNA structures and for sequences that allow an otherwise inert plasmid to replicate in the yeast Saccharomyces cerevisiae. Analysis of one of these clones, ARSH1, showed that the minimal region showing this origin activity in yeast cells contains modular elements similar to those present in the best characterized yeast replicator, ARS1. Recently we have investigated the abundance of nascent DNA strands around a 30 kb human DNA region encompassing the ARSH1 locus, by both conventional and real-time PCR. Our results indicate that putative initiation sites for DNA replication are present in the DNA region encompassing the ARSH1 sequence. Based on these results, the major goals of this research proposal are: (i) to identify potential origins of DNA replication present within a 30kb DNA region encompassing ARSH1; (ii) to determine the binding of putative origins around ARSH1 to proteins required for initiation of DNA replication in human cells; (iii) to test if putative origins around ARSH1 function at ectopic locations, and to dissect the DNA elements required for their activity. It is anticipated that these studies will provide a better description of human origins of replication, and increase our knowledge about the mechanism that leads to the activation of specific replicons. An understanding of these areas may also provide new targets for developing anti-proliferative drugs that could be used to treat cancer.